20 results about "Calcifediol" patented technology

The invention relates to the technical field of agricultural biology, in particular to a fusion P450 enzyme mutant, a method for improving VD3 catalytic activity and a method for generating 25 (OH) VD3 through whole cells. The invention constructs a fusion P450 enzyme VK1-CYP116B46-L21 and a self-sufficiency fusion P450 enzyme mutant with improved activity, wherein the self-sufficiency fusion P450 enzyme mutant is used for catalyzing vitamin D3 to synthesize calcifediol; vD3 is taken as a substrate, glucose, a cosolvent and a wet thallus induced by recombinant escherichia coli for simultaneously expressing fusion P450 enzyme and glucose dehydrogenase are added, and the highest yield of 25 (OH) VD3 within 24 hours after catalytic reaction can reach 3.26 g / L; whole cells are directly used as a catalyst, enzyme purification is not needed, glucose dehydrogenase is introduced, and glucose is used as a substrate to regenerate a cofactor NAD (P) H, so that addition of expensive cofactors is avoided, the production cost is reduced, and industrial production is facilitated.

The invention relates to the technical field of biology, in particular to a pichia pastoris recombinant strain for expressing non-specific peroxygenase CciUPO and application of the pichia pastoris recombinant strain. The pichia pastoris recombinant strain is obtained by the following steps: S1, synthesizing a sequence of a CciUPO original gene sequence optimized based on a yeast codon; s2, carrying out recombination with the EV signal peptide after the directed evolution; s3, connecting the recombined target gene with an expression vector to construct a recombinant plasmid; and S4, transforming the recombinant plasmid into pichia pastoris competent cells, and screening out a recombinant strain containing the CciUPO recombinant gene. The expression quantity of the CciUPO obtained through the operation is increased by 10 times compared with that of a wild type CciUPO, the CciUPO has strict catalytic regioselectivity, can catalyze vitamin D3 to synthesize calcifediol, the selectivity is as high as 95%, the catalytic conversion rate of toluene is 95%, the selectivity is 78%, and the CciUPO has a relatively high application prospect in industrial production.

The application belongs to the technical field of bio-chemical industry, and particularly relates to a P450 enzyme mutant and application of an engineering bacterium thereof in synthesis of calcifediol, the mutant is one or more point positions in an amino acid sequence based on a wild type P450 enzyme Vdh being mutated; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to GenBank accession number CP069288.1. Through systematic molecular dynamics simulation, conservation analysis and substrate binding pocket engineering on the Vdh enzyme, multiple key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I114R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, and the pure enzyme activity can reach 9.8 times of the wild type enzyme, greatly improving the conversion rate from vitamin D3 to calcifediol.

The invention belongs to the technical field of biological catalysis, and discloses a non-specific peroxygenase mutant, a preparation method and application thereof, and a preparation method of calcifediol. Compared with a wild type non-specific peroxygenase (comprising an amino acid fragment with a sequence as shown in SEQ ID NO: 1), the non-specific peroxygenase mutant provided by the invention has one or more of Y47L mutation, L83F mutation and V112E mutation. The non-specific peroxygenase mutant has excellent enzyme activity, has the advantages of high conversion rate, mild reaction conditions, low cost and the like when being applied to preparation of calcifediol, and has a good industrial application prospect.

The present invention discloses a P450 enzyme mutant, a single-plasmid three-enzyme co-expression system, and their application in calcifediol synthesis. The P450 enzyme mutants include: using the wild-type P450 enzyme CYP109E1 shown in SEQ ID NO. 1 as a template, and respectively mutating the T at position 78 or the G at position 81 to form the mutants T78A, T78V, T78L, and T78I, G81V, and G81M; and mutating both the T at position 78 and the G at position 81 to form the mutant T78L / G81M. The present invention also constructs a single-plasmid three-enzyme co-expression system of the P450 enzyme mutant and a redox partner, thereby improving the electron transfer efficiency in the P450 enzyme hydroxylation reaction. This system can synthesize calcifediol with higher efficiency under mild conditions, solving the problems of low catalytic efficiency and activity in the synthesis of calcifediol catalyzed by P450 enzymes, and has important industrial application value.

The invention belongs to the technical field of biochemical engineering, and particularly relates to application of a P450 enzyme mutant and engineering bacteria thereof in synthesis of calcifediol, and the mutant is obtained by mutating one or more sites in an amino acid sequence based on a wild type P450 enzyme Vdh; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to the accession number of GenBank, namely CP069288.1. Through systematic molecular dynamics simulation, conservative analysis and substrate binding pocket engineering on the Vdh enzyme, a plurality of key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I144R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, the pure enzyme activity of the combined mutant can reach 9.8 times that of a wild type enzyme, and the conversion rate from vitamin D3 to calcifediol is greatly increased.

Disclosed herein is an improved, cost-effective scalable process for preparation of 25-OH 7-DHC ( 25-OH provitamin D3(Calcifediol).

The invention discloses a method for recovering mother liquor after purification of crude products of vitamin D3 or metabolic derivatives thereof. Comprising the following steps: evaporating mother liquor obtained by purifying a crude cholecalciferol or calcifediol product to remove a solvent so as to obtain an oily concentrate, adding the oily concentrate into high-boiling-point grease, uniformly mixing, and collecting and recovering cholecalciferol or calcifediol by using a molecular distillation technology. According to the method, the molecular distillation technology is utilized to efficiently recover the cholecalciferol or the calcifediol, so that the content of the cholecalciferol or the calcifediol in the cholecalciferol or the calcifediol resin oil is increased to 80% or above, the recovery rate of the cholecalciferol or the calcifediol is 90% or above, the process is simple, the flow is short, the processing process is a physical separation process, and the method is suitable for industrial production. And no chemical substance harmful to the human body is added.

The invention discloses a calcifediol spray drying tower air inlet temperature control device and method, and relates to the technical field of calcifediol production equipment.The calcifediol spray drying tower air inlet temperature control device comprises an air inlet pipe, a plurality of heating pipes are arranged in the air inlet pipe at equal intervals, and a plurality of temperature sensors are fixedly arranged on the surface of the air inlet pipe; the temperature sensors are evenly distributed in the air inlet pipe in the circumferential direction and the axial direction of the air inlet pipe, and inductive probes of the temperature sensors penetrate through the air inlet pipe and extend into the air inlet pipe. The device has the beneficial effects that the plurality of temperature sensors which are uniformly distributed in the circumferential direction and the axial direction of the air inlet pipe and of which the sensing probes extend into the air inlet pipe can comprehensively detect the air temperature at different positions in the air inlet pipe, and compared with a single sensor, more accurate and comprehensive temperature data can be obtained, a reliable basis is provided for subsequent temperature regulation and control, and the device is suitable for popularization and application. The accuracy of inlet air temperature detection is effectively improved, and then the temperature control precision is improved.

The present invention discloses a genetically engineered bacterium, which is an engineered bacterium constructed by expressing CYP gene, ferredoxin gene, ferredoxin reductase gene and dehydrogenase gene in cells; wherein, the amino acid sequence encoded by the CYP gene is as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.9 or SEQ ID NO.11. The present invention also discloses the application of the genetically engineered bacterium in the preparation of calcifediol and / or calcitriol. When the engineered bacterium of the present invention is applied to the synthesis of calcifediol and calcitriol, the yields are significantly improved. When it is applied to industrial production, the cost is lower, and the reaction has high specificity, mild reaction conditions and environmental friendliness, meeting the industrial production requirements of calcifediol and calcitriol.

The present invention discloses novel method for synthesizing vegan 25-OH cholesterol / Calcifediol from inexpensive crude phytosterol. According to the method, Phytosterols are reacted to form corresponding i-steroid through tosylation and methanolysis. i-steroid on reductive ozonolysis to C-22 alcohol and conversion via C-22 tosylate to C-22 iodide in good yield. Coupling of C-22 tosylate with Grignard reagent of 4-bromo-2-methyl-2-[(trimethylsilyl)oxy] butane followed by deprotection yielded 25-OH cholesterol. In a process variant, nickel mediated conjugate addition of C-22 iodide to an electron deficient alkene ethyl acrylate and treating corresponding ester with methyl magnesium bromide as means of installing the side chain of 25-OH cholesterol in high yield. Further bromination reaction of 25-OH cholesterol diacetate followed by dehydrobromination using TBAF yielded 25-OH 7-dehydrocholesterol. Further photo reaction of 25-OH 7-dehydrocholesterol in to previtamin D3 using high or medium pressure mercury lamp and subsequent thermal reaction of previtamin D3 to 25-OH vitamin D3(Calcifediol) in good yield.

The invention belongs to the technical field of biochemical engineering, and particularly relates to a P450 fusion protein, the structure of the fusion protein is VdhM3-Linker1-AciC-Linker2-AciB, VdhM3 is Vdh-M3 (I144R / N173M / Q310R), and VdhM3 is Vdh-M3 (I144R / N173M / Q310R); the GenBank login numbers of the coding genes of the AciC and the AciB are both AB221118.1, and the GenBank login numbers of the coding genes of the AciC and the AciB The amino acid sequence of the Linker2 is GSGSGH; and the Linker 1 is a flexible connector, a rigid connector or a flexible-rigid combined connector. By means of system screening, it is determined that the AciC / AciB redox partner system derived from acinetobacter has the optimal suitability with VdhM3 P450 enzyme, the catalytic activity is high, the selectivity of the target product calcifediol reaches up to 95%, generation of by-products is reduced, and the purification efficiency and yield of the product are improved.

The application discloses a method for extracting calcifediol from a crystallization mother liquor of a calcifediol crude product. The method uses a crystallization technique to extract the crystallization mother liquor of the calcifediol crude product obtained through a photochemical reaction for a second time to obtain a calcifediol crystal with a higher content, and then through a recrystallization process, a crystalline pure product meeting product quality standards is obtained. The specific method is as follows: after the solvent of the mother liquor after purification of the calcifediol crude product is evaporated, an oily concentrate is obtained, the oily concentrate is dissolved with an organic solvent, then activated carbon is added to decolorize at a certain temperature, filtration is performed, cooling crystallization is performed, filtration is performed, the solid is dissolved with an organic solvent for a second time, cooling crystallization is performed, filtration is performed, and drying is performed to obtain a finished product. The application has the advantages of simple process, short flow, high efficiency and high product purity.

The present disclosure relates to a combination of active ingredients / adjuvants for use in the treatment of nervous system disorders and diseases such as Alzheimer's disease and mild cognitive impairment (MCI) and memory and cognitive impairments and disorders. In particular, scyllo-inositol and a combination for the treatment of Alzheimer's disease, such as Arcamikumab, and / or a combination with an essential fatty acid, such as a mixture of linolenic acid / linoleic acid, or vitamin D or a vitamin D compound, such as calcifediol, are useful. The combination may be in the form of a separate dosage form for each active ingredient, or may be in an oral dosage form with multiple active ingredients in a single capsule or tablet or oral solution. The present invention also relates to a method of treating a patient suffering from a mild cognitive disorder having an MMSE standard between 22 and 26 with a pharmaceutically effective amount of scyllo-inositol to treat said disease and slow progression to Alzheimer's disease.

The application belongs to the technical field of bio-chemical industry, and particularly relates to a P450 fusion protein, and a structure of the fusion protein is VdhM3-Linker1-AciC-Linker2-AciB, wherein VdhM3 is Vdh-M3 (I114R / N173M / Q310R); the GenBank accession numbers of the encoding genes of AciC and AciB are both AB221118.1; the amino acid sequence of Linker2 is GSGSGH; and Linker1 is a flexible linker, a rigid linker or a flexible-rigid combined linker. Through systematic screening, it is determined that the AciC / AciB oxidation-reduction partner system derived from Acinetobacter has the best adaptability with the VdhM3 P450 enzyme, the catalytic activity is high, and the selectivity of the target product calcifediol is as high as 95%, the generation of by-products is reduced, and the purification efficiency and yield of the product are improved.

Disclosed herein is an improved and efficient process for synthesis of vitamin D3 and its analogue Calcifediol from Ergosterol. Particularly, the present invention discloses the synthesis of key intermediate 3β-tert-Butyldimethylsilyloxy-22-hydroxy-23,24-bisnorchola-5,7-diene (5), and novel intermediate β-tert-Butyldimethylsilyloxy-22-iodo-23,24-bisnorchola-5,7-diene (9) by a simple and cost effective process. The industrially viable processes for preparation of said intermediate(s) results in providing provitamins with various side chains and the desired products in high yield.

The application discloses a P450 mutant and application thereof in synthesis of calcifediol, and belongs to the technical field of bioengineering. The P450 mutant enzyme is constructed, and a single-plasmid three-enzyme co-expression system of an oxidation-reduction partner and a cofactor circulation enzyme is constructed, so that sufficient electron transfer chains are provided for the P450 enzyme, and calcifediol can be synthesized with higher efficiency under mild conditions and with vitamin D3 as a substrate. The conversion rate of the calcifediol can reach 63.1%, and the conversion rate of the reaction is greatly improved. The method disclosed by the application improves the catalytic efficiency of the catalyst and reduces the cost of the reaction.

The invention discloses a high-concentration 7-dehydrocholesterol enzymatic hydroxylation method based on an organic-water two-phase system, which comprises the following steps: loading high-concentration 7-DHC (7-dehydrocholesterol) through an organic phase, maintaining peroxygenase activity through a water phase, and catalyzing 25-site hydroxylation of 7-DHC by taking hydrogen peroxide as an oxygen source to generate 25-hydroxy-7-dehydrocholesterol. According to the method, the problem of low solubility caused by hydrophobicity of 7-DHC is solved, the substrate concentration is greatly improved, the conversion rate and the product selectivity are improved, the method is suitable for industrial production of calcifediol, the productivity can be improved, and the cost can be reduced.

The invention belongs to the technical field of biological catalysis, and particularly discloses a method for synthesizing calcifediol by using a photocatalytic device. According to the method, 7-dehydrocholesterol is used as a substrate, and 25-hydroxy-7-dehydrocholesterol is obtained through the steps of non-specific oxide enzyme catalysis, extraction, reduced pressure distillation and the like. Dissolving the 25-hydroxy-7-dehydrocholesterol in a polar solvent, a non-polar solvent or a mixed solvent system of the polar solvent and the non-polar solvent, adding an anti-oxidation reagent, and carrying out an illumination isomerization reaction; and under the protection of inert gas, carrying out thermal isomerization reaction on the reaction liquid after photochemical treatment, then carrying out low-temperature crystallization to obtain part of calcifediol crystals, collecting the filtrate after crystallization, and carrying out thermal isomerization reaction and low-temperature crystallization for multiple times to obtain the residual calcifediol crystals. The method has the advantages of simple operation steps, small usage amount of the organic solvent, high synthesis efficiency and cost saving, and is beneficial to large-scale industrial production of calcifediol.